Enhanced Energy Expenditure and Fat Oxidation in Humans with High BMI Scores by the Ingestion of Novel and Non-Pungent Capsaicin Analogues (Capsinoids)

60341 (048)

Biosci. Biotechnol. Biochem., 71, 60341-1–10, 2007

y Naohiko INOUE, Yoshiko MATSUNAGA, Hitoshi SATOH, and Michio TAKAHASHI

Ajinomoto Research Institute for Health Fundamentals, 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan

Received June 20, 2006; Accepted October 15, 2006; Online Publication, February 7, 2007 [doi:10.1271/bbb.60341]

The biochemical and physiological indices were energy expenditure in a weight-loss program together monitored in 44 subjects after 4-week capsinoids with reducing the energy intake (‘‘dieting’’). (capsaicin analogues with low pungency) intake. The According to the National Nutritional Survey in Japan subjects were randomly assigned to 3 groups: CSNs3 between 1988 and 2004,3) the mean body weight of the (3 mg/kg of capsinoids), CSNs10 (10 mg/kg of capsi- Japanese populace was continuously increasing in those noids) and the control (placebo). Measurements were years, although the dietary intake tended to decrease and performedAdvance in the morning on overnight-fasted View subjects. habitual physical activities remained unchanged. This The oxygen consumption (VO2), resting energy expen- continual increase in body weight could be attributable diture (REE) and fat oxidation increased slightly to the attenuation of BMR with age. BMR constitutes a compared to pre-administration values without any major share (60–70%) of the daily energy expenditure adverse effects, although the increase was not signifi- and has been documented to decrease with age4–10) and cant. The increase in fat oxidation was positively and with a high body mass index (BMI).11,12) significantly correlated with the body mass index (BMI). Capsaicinoids have been well documented as sub- A meta-analysis was therefore conducted on a subgroup stances for increasing BMR. For example, in a human consisting of subjects with BMI 25 (n ¼ 28). As a trial where capsaicin at a relatively high-dose was result, not only VO2 increased significantly (p < 0:05)in ingested for 3Proofs months, increased fat oxidation was the CSNs10 group, but also REE in the CSNs10 group reported to be sustained together with sustained eleva- and fat oxidation in the CSNs3 and CSNs10 groups tion of the resting energy expenditure (REE).13) How- tended to increase (p < 0:1). Consequently, a capsinoids ever, capsaicin has such strong pungency and nocicep- intake would be able to enhance the energy expenditure tive stimulus that not all people can eat much and avoid and fat burning in humans, particularly those with high a stomach ache. Capsinoids including ‘capsiate’ are non- BMI. pungent capsaicinoid analogues and are derived from a non-pungent cultivar of Capsicum annuum L. (‘CH-19 Key words: ‘capsiate’; capsinoid; resting energy expen- sweet’). The non-pungent capsinoid known as ‘capsiate’ diture; fat oxidation; body mass index is known to have similar physiological effects to those of capsaicin: a single oral administration of ‘capsiate’ It is of utmost importance to improve obesity for has been reported to increase the oxygen consumption 14,15) 16) health maintenance in life. Obesity is the result of the (VO2) and body temperature in mice like cap- energy intake exceeding the energy expenditure.1) It is saicin, reflecting increased energy expenditure. A thus necessary to either reduce the energy intake or chronic (2-week) administration of ‘capsiate’ to mice enhance the energy expenditure to resolve the problem has increased the 24-hr total VO2, fat tissue uncoupling of obesity. However, it is not easy to attain the weight protein (UCP-1) and its mRNA associated with a loss only by reducing the energy intake because of an reduction in the fat tissue weight and body weight.17) increasing sense of hunger or a risk of attenuation in the As for human studies, there is a report describing basal metabolic rate (BMR). The latter often causes one enhanced VO2 and body temperature in volunteers of the reasons for weight regain.2) Therefore, it is immediately after a single intake of ‘CH-19 sweet’ advantageous to include certain strategies to enhance the fruit.18) This present study was designed to assess the

y To whom correspondence should be addressed. Fax: +81-44-244-0495; E-mail: naohiko [email protected] Abbreviations: CSNs3, group ingesting 3 mg/day of capsinoids; CSNs10, group ingesting 10 mg/day of capsinoids; BMI, body mass index; VO2, oxygen consumption; REE, resting energy expenditure; RQ, respiratory quotient; BMR, basal metabolic rate; FFM, fat free mass; BAT, brown adipose tissue; UCP, uncoupling protein; SNS, sympathetic nervous system; TG, triglyceride; FFA, free fatty acid; MCT, medium-chain triglyceride 60341-2 N. INOUE et al. change in energy metabolism after a long-term intake of For a comparison of their potency, three synthetic capsinoids. To avoid any acute capsinoid effects, various compounds were assessed for their potency toward parameters were monitored approximately 24 hours after calcium uptake, adrenalin secretion with i.v. adminis- the last ingestion of capsinoids during the course of a 4- tration and change in VO2 with intragastric adminis- week ingestion study. In addition, the extracted oil of tration. Each capsinoid elicited virtually similar effects ‘CH-19 sweet’ fruit was prescribed in this study. (unpublished data), so the content of capsinoids was Obese or obesity-prone subjects with BMI > 23 were expressed as the total amount of capsinoids. recruited (the Japan Society for the Study of Obesity has Apart from capsinoids, ‘CH-19 sweet’ extracted oil defined BMI 25 as obese and BMI = 22.2 as standard contained small amounts of MCT (0.019 or 0.064 g due weight). REE has been reported to be somewhat to different batches) and capsaicin (0.0020 or 0.0067 dependent on the BMI value; a decrease in REE mg). Qualitatively, these contents would affect the adjusted for the fat-free mass (FFM) was accompanied energy metabolism,13,22–25) but the amounts used in the with a BMI increase,12) and an increase in the attenu- present study seem to have been too small to exert any ation rate of sleep energy expenditure was accompanied biological effect based on the effective amount reported with a BMI increase.19) Thus, we investigated not only in published documents. the effect on resting energy expenditure by a long-term intake of capsinoids, but also whether the effects of Protocol. The three groups were compared in a capsinoids depended on the BMI values. randomized double-blind study over a 4-week period. Each group ingested 5 capsules; for CSNs3, there were Materials and Methods three capsules each containing 1 mg of capsinoids and two capsules of the placebo. The dose was temporarily Subjects. Men and postmenopausal women (30–65 set as ‘‘normal dose (3 mg)’’ or ‘‘high dose (10 mg)’’ yearsAdvance old) with a BMI score higher than 23 kg/m View2 were based on a previous observation that 2.1–7.0 mg/day of recruited from the Soiken Clinic databank in Osaka. capsiate significantly increased VO2 in human sub- Those who had abnormal clinical laboratory test values, jects.18) The respiratory gas, body weight, height, hepatopathy, nephropathy, cardiovascular disturbance, diastolic and systolic blood pressure and pulse rate were breathing disorder, endocrinopathy, defective metabo- monitored at 0 (before ingestion), 2 and 4 weeks post- lism, gastric ulcers, allergic symptoms to red pepper, or treatment. Blood samples were also taken for hemato- under medical treatment for obesity and hyperlipemia logical and blood biochemical analyses. All the subjects were excluded. Men with waist < 85 cm/height > 170 underwent the study as a single sample population at the cm, and those with waist < 80 cm/height < 170 cm Soiken Clinic with analyses conducted under the super- were excluded. The remaining 48 subjects (39 males and vision/managementProofs of the designated clinician. 9 females) were randomly assigned to the CSNs3 group Three days before the start, the subjects were receiving daily 3 mg/day of capsinoids, CSNs10 group instructed to refrain from snacks and alcoholic bever- receiving 10 mg/day of capsinoids or the placebo ages. From one day before the start, the subjects took (control) group. Four subjects withdrew for personal menu-designated meals 3 times a day and were asked to reasons, leaving 44 eligible subjects: CSNs3 (n ¼ 14), take supper by 21:00 hr (no eating nor drinking there- CSNs10 (n ¼ 15) and controls (n ¼ 15). The subjects after), so that the subjects fasted overnight for more than were briefed on the purpose and the outline of this study, 12 hr. Details of the meals are as follows: heat energy and signed a consent form before the study. This study was 500, 735 and 765 kcal for breakfast, lunch and was approved by the Institutional Review Board for supper, respectively; protein, fat, carbohydrate and salt studies of food in humans by Ajinomoto Co., Inc. were 25, 10, 70 and 3.5 g for breakfast, 35, 22, 90 and (Tokyo, Japan) and by the Joint Institutional Review 3.5 g for lunch, and 40, 23, 90 and 3.5 g for supper, Board of Soiken, Inc. and Soiken Clinic (Osaka, Japan), respectively. On the day for monitoring the data already in accordance with the concept of the Helsinki Decla- mentioned, the subjects were asked not to take any meal, ration. smoke or do physical exercise after being awaken but up to 350 ml of drinking water was allowed until 2 hr before Test sample. The sample was oil extracted from ‘CH- monitoring. In addition to the test-meal intake, the 19 sweet’ fruit containing capsinoids. Dried fruits from subjects were asked not to change their normal eating, ‘CH-19 sweet’ were extracted with hexane and treated smoking and physical exercise habits. with medium-chain triglyceride (MCT) before refining by evaporation and condensation. The extracted oil Respiratory gas analysis. The resting energy expen- diluted with canola oil was sealed in marigold-colored diture (REE) was measured by the breath-by-breath soft capsules containing 1 or 2 mg of capsinoids. method, using a respiratory gas analyzer (AE-300S; Capsules not containing the extracted oil were used as Minato Medical Science, Osaka, Japan). The measure- a placebo. ments were carried out for 20 min after overnight The capsinoids consisted of three different forms: fasting. Each subject sat quietly for 30 min and then ‘capsiate’, dihydrocapsiate and nordihydrocapsiate.20,21) for 10 min with a face mask before measurement in the Enhanced Energy Expenditure by Capsinoids in Humans 60341-3 sitting position. The data obtained during the last 10 min treatment were made with the Bonferroni multiple- were analyzed. The resting energy expenditure (REE), comparison test. In respect of the hematological and respiratory quotient (RQ), glucose oxidation and fat biochemical data, comparisons were made between the oxidation were calculated by the VO2 and carbon groups with the Dunnett multiple-comparison test. All dioxide production. the values obtained at 0 week were compared with the corresponding values obtained at 4 weeks with Student’s Body weight, height, BMI, blood pressure, pulse rate t-test. Statistical software (SPSS Ver. 11.5, SPSS Co., and medical examination. All the subjects underwent Ltd.) was employed in the respective comparisons, measurements of body weight, standing height, blood where a risk factor of p < 0:05 in the paired testing was pressure, and pulse rate, and a medical examination at considered statistically significant. the Soiken Clinic. The subjects were requested to visit the clinic without taking the test sample capsules. The Results blood pressure and pulse rate were measured in a sitting position after a 10-min rest. All medical examinations Respiratory gas analysis were performed by the designated medical doctor. BMI The results of the respiratory gas analysis are shown was calculated as the body weight (kg) divided by the in Table 1 and Fig. 1. No significant inter-group dif- square of the height (m2). ferences were observed in any parameters (Table 1). However, changes in VO2, REE, RQ, glucose oxidation Blood test. Blood samples were taken in the clinic and fat oxidation were slightly higher in the capsinoids- from all the subjects after overnight fasting, and the treated groups (CSNs3 and CSNs10), though the following measurements were taken using peripheral increases were not significant. Furthermore, CSNs3 blood samples: blood properties (leucocyte count, and CSNs10 showed a lower mean value for the glucose erythrocyteAdvance count, hemoglobin, hematocrit, View mean cor- oxidation level together with a higher mean value for fat puscular volume, mean corpuscular hemoglobin, mean oxidation level than the controls (Fig. 1), although the corpuscular hemoglobin concentration, platelet count), differences were not significant. aspartate aminotransferase, alanine aminotransferase, To discover a more responsive subgroup by a meta- lactate dehydrogenase, -glutamyltranspeptidase, total analysis, we inspected the correlations between every bilirubin, total protein, albumin, alkaline phosphatase, above-mentioned parameter and the BMI value at 0 urea nitrogen, uric acid, creatinine, electrolytes (Naþ, week. The BMI value tended to be correlated with the Kþ,Cl,Ca2þ, and Mg2þ), blood glucose, insulin, fat oxidation increase of either CSNs3 or CSNs10 (R ¼ hemoglobin A1c, total ketone body, 3-hydroxybutyric 0:424, p ¼ 0:131 or R ¼ 0:447, p ¼ 0:109), and was acid, acetoacetic acid, total cholesterol, HDL-cholester- correlated wellProofs with the combined fat oxidation in- ol, LDL-cholesterol, RLP-cholesterol, TG, free fatty creases in both CSNs3 and CSNs10 (R ¼ 0:434, p ¼ acid (FFA) and phospholipids. 0:018) (Fig. 2), suggesting that the higher-BMI subjects Blood sampling was performed after a 10 min rest in a would respond more to capsinoids by enhancing fat sitting position. All measurements were taken by SRL oxidation. We then tentatively introduced the BMI Co., Ltd., and Osaka Serum Microbiology Laboratories 25 kg/m2 criterion for making a subgroup that would Co., Ltd., according to appropriate methods. presumably more responsive. BMI 25 kg/m2 is a standard criterion for obesity described by the Japan Recording the daily diet and number of walking steps. Society for the Study of Obesity. The meta-analysis on The subjects were asked to record either the dietary the population (a subgroup) with BMI 25 kg/m2 gave contents to confirm they had kept to their normal eating a significant VO2 increase between 2 weeks and 0 week routines or to utilize a record for nutritional analysis. (p < 0:05) in CSNs3, with an increasing tendency (p < Dieticians calculated the nutritional ingredient intake 0:1) for REE (Table 2). Fat oxidation at 0 week tended (energy, protein, fat, carbohydrate, cholesterol, and to be lower in CSNs3 than in the controls (p < 0:1). th dietary fiber) based on the 5 Revision of the Japan Moreover, VO2 was significantly higher (p < 0:05)in Standard Food Ingredient Table. The numbers of daily CSNs10, with an increasing tendency of REE and fat walking steps were recorded with a pedometer before oxidation (p < 0:1), when compared with the controls the day when the subject visited the clinic at 0, 2 and 4 (Fig. 3). Fat oxidation in CSNs3 also tended to be higher weeks post-treatment. than that in the controls (p < 0:1). There were no significant changes in VO2, REE, RQ, glucose oxidation Statistical analyses. Each value is expressed as the and fat oxidation in the control group throughout the mean standard error. The respiratory gas analysis, measurements. body weight, BMI, diastolic and systolic blood pressure, pulse rate, nutrient intake and number of walking steps Body weight, BMI, blood pressure, pulse rate and were compared between the groups by the Dunnett medical examination multiple-comparison test. In addition, comparisons in Data on the body weight, BMI, diastolic and systolic each group among the data at 0, 2 and 4 weeks post- blood pressure, and pulse rate are summarized in 60341-4 N. INOUE et al.

Table 1. VO2, REE, RQ, Fat Oxidation and Glucose Oxidation Values before and after the Intake of Capsinoids

week 024 CSNs10 3:12 0:09 3:18 0:09 3:15 0:07 VO 2 CSNs3 3:13 0:06 3:21 0:08 3:17 0:08 (ml/min/kg) controls 3:21 0:07 3:23 0:09 3:21 0:08 CSNs10 21:4 0:59 21:8 0:62 21:6 0:52 REE CSNs3 21:5 0:41 22:0 0:55 21:8 0:58 (kcal/day/kg) controls 22:0 0:49 22:2 0:58 22:1 0:52 CSNs10 0:78 0:01 0:79 0:01 0:78 0:01 RQ CSNs3 0:79 0:01 0:79 0:01 0:79 0:01 controls 0:78 0:01 0:80 0:01 0:79 0:01 CSNs10 80:6 4:782:4 5:983:4 5:1 Fat oxidation CSNs3 78:5 3:582:2 4:581:0 5:2 (mg/min) controls 87:4 5:281:9 5:880:7 3:6 CSNs10 71:3 13:077:6 12:971:3 11:6 Glucose oxidation CSNs3 80:6 10:477:2 8:277:2 10:1 (mg/min) controls 71:2 9:185:8 10:886:4 10:3

Each values is the mean SE. CSNs10, n ¼ 15; CSNs3, n ¼ 14; controls, n ¼ 15.

AdvanceA ViewB 0.3 2.0 0.2 1.5 1.0 0.1 0.5 0.0 CSNs10 (ml/min/kg)

2 0.0 -0.1 CSNs3

VO -0.5

∆ REE (kcal/day/kg) controls -0.2 ∆ -1.0 024 024 week week C D E 0.82 30 Proofs20 20 15 0.80 10 10 5 0.78 0 0 -10 (mg/min) (mg/min) -5

0.76 oxidation Fat -20 ∆ -10 Glucose oxidation 0.74 ∆ -30 -15 Respiratory quotient 024 024 024 week week week

Fig. 1. Changes in (A) Oxygen Consumption (VO2), (B) Resting Energy Expenditure (REE), (C) Respiratory Quotient (RQ), (D) Glucose Oxidation and (E) Fat Oxidation from the Initiation of Sample Ingestion. Respiratory gas analyses were performed on overnight-fasted subjects without taking the test sample in the morning. Each value is expressed as the mean SE. (CSNs3, n ¼ 14; CSNs10, n ¼ 15; controls, n ¼ 15).

Table 3. The body weight and BMI of CSNs3 and Although a significant weight loss could be expected CSNs10 were tended to decrease during the 2 to 4 week in the subgroup with BMI 25 kg/m2, that was not the period but significant differences were not observed case (data not shown). The systolic blood pressure had between any two of the three groups. Significant increased significantly by week 4 in CSNs10 (data not differences in the diastolic and systolic blood pressure shown), although the increase value was still within the were not apparent between any two of the three groups. normal range. An increasing trend was observed in the pulse rates of CSNs3 and CSNs10 through the 2 to 4 week period, Blood examination although the increases were within the normal range. In The blood parameters related to the carbohydrate and addition, the supervising physician did not extract any fat metabolism demonstrated no significant inter-group causal effect of capsinoids ingestion from individual differences. Significantly higher in insulin (p < 0:05) interviews. and a tendency to higher blood glucose (p < 0:1) and Enhanced Energy Expenditure by Capsinoids in Humans 60341-5

40 significant differences in the lactodehydrogenase level 30 between CSNs10 and the control at 0 and 4 weeks. As 20 this difference existed before the treatment started, there 10 would not have been any clinical implications. No 0 significant inter-group differences were apparent in the other parameters. There were some significant inter- (mg/min) -10 Fat oxidation Fat

∆ period differences throughout the three groups in such -20 parameters as hematocrit, mean corpuscular volume, -30 mean corpuscular hemoglobin, mean corpuscular hemo- 22 24 26 28 30 32 34 2 globin concentration, alanine aminotransferase, -gluta- BMI (kg/m ) myltranspeptidase, total bilirubin, albumin, alkaline phosphatase, urea nitrogen, uric acid, Kþ,Cl,Ca2þ, Fig. 2. Combined Correlations between the Increases in Fat Oxida- 2þ tion at 4 Weeks Post-Treatment and BMI at 0 Week Post-Treatment and Mg . All differences, however, were within the (n ¼ 29) in CSNs3 (3 mg/kg of capsinoids) and CSNs10 (10 mg/ normal range. No marked differences, indicating abnor- day of capsinoids). malities in hepatic and renal functions, were not Fat oxidation is expressed as the increase from 0 to 4 weeks post- apparent. p ¼ : treatment. The correlation coefficient is 0.4363 ( 0 018). A gender-differentiated analysis and analysis of the subgroup with BMI 25 kg/m2 were also carried out. FFA levels (p < 0:1) were observed in CSNs10 between Although there were significant inter-group or inter- weeks 0 and 4. A tendency to higher phospholipids period differences in some parameters (data not shown), (p < 0:1) in CSNs3 and a significant increase in blood all differences were small in absolute terms and within glucose level (p < 0:01) in the controls were observed the normal range, suggesting they did not pose any betweenAdvance weeks 0 and 4. Viewclinical problems. The HDL-cholesterol level had a gender difference, so the data were analyzed separately. However, no differ- Daily diets and number of walking steps ence was apparent between any two of the three groups. The daily nutrient intake and number of walking steps When focused on the subgroup with BMI 25 kg/m2, were examined before the day when the subjects the insulin level in CSNs3 tended to be lower than the underwent monitoring at the clinic (Table 4). Any control level at 4 weeks (p < 0:1; data not shown). The significant inter-group differences were observed. While total ketone body and acetoacetic acid levels in CSNs3 the protein intake tended to increase in CSNs10 between tended to be higher than the control values at 4 weeks 0 and 4 weeks, the variation was within the normal (p < 0:1). The control values at 4 weeks indicated a range, implying that the influence would be negligible. Proofs2 decreasing tendency for the total ketone body level (p < In the subgroup with BMI 25 kg/m , the intake of 0:1) and a significantly decreasing 3-hydoxybutyric acid cholesterol was significantly less in CSNs10 than in the level (p < 0:05; data not shown). control (p < 0:05), although the variation was within the The hematological and biochemical data only yielded normal range (data not shown).

2 Table 2. VO2, REE, RQ, Fat Oxidation and Glucose Oxidation Values before and after the Intake of Capsinoids in Subjects with BMI 25 kg/m

week 02 4 CSNs10 3:09 0:09 3:23 0:10 3:17 0:08 VO 2 CSNs3 3:10 0:06 3:22 0:08 # 3:16 0:09 (ml/min/kg) controls 3:21 0:09 3:19 0:08 3:15 0:08 CSNs10 21:2 0:64 22:1 0:63 21:8 0:57 REE CSNs3 21:3 0:44 22:1 0:57 + 21:7 0:65 (kcal/day/kg) controls 22:0 0:57 21:9 0:51 21:7 0:51 CSNs10 0:80 0:01 0:79 0:01 0:78 0:01 RQ CSNs3 0:80 0:01 0:79 0:01 0:79 0:01 controls 0:78 0:01 0:80 0:01 0:80 0:01 CSNs10 80:0 3:391:2 5:591:3 4:0 Fat oxidation i CSNs3 78:9 3:2 85:6 3:1 + 84:5 4:6 (mg/min) y controls 92:6 4:386:4 5:684:1 3:7 CSNs10 92:8 12:986:1 13:980:8 12:6 Glucose oxidation CSNs3 89:5 9:182:0 8:079:9 9:0 (mg/min) controls 80:6 8:592:4 13:595:3 11:9

Each value is the mean SE. CSNs10, n ¼ 8; CSNs3, n ¼ 11; controls, n ¼ 9.+,#:p < 0:1, 0.05 vs. baseline by the Bonferroni test. y: p < 0:1 between 2 groups by the Dunnett test. 60341-6 N. INOUE et al.

A B 0.3 (A)2.0 (B) * † 0.2 * 1.5 † # 1.0 0.1 + 0.5 0.0 CSNs10 (ml/min/kg)

2 0.0 CSNs3 -0.1 -0.5 VO REE (kcal/day/kg) ∆ controls -0.2 ∆ -1.0 024 024 C week D week E 0.82 (C)30 (D)20 (E) 20 15 0.80 † † 10 10 5 0.78 0 + † 0 † -10 (mg/min) (mg/min) -5 0.76 oxidation Fat -20 ∆ -10 Glucose oxidation 0.74 ∆ -30 -15 Respiratory quotient 024 024 024 week week week

Fig. 3. Change in (A) Oxygen Consumption (VO2), (B) Resting Energy Expenditure (REE), (C) Respiratory Quotient (RQ), (D) Glucose Oxidation and (E) Fat Oxidation from the Initiation of Sample Ingestion in Those Subjects with BMI 25 kg/m2. Respiratory gas analyses were performed on overnight-fasted subjects without taking the sample in the morning. Each value is expressed as Advancethe mean SE. Subjects with BMI 25 kg/m2 were View assigned to 3 groups: CSNs3 (3 mg/kg of capsinoids, n ¼ 11), CSNs10 (10 mg/day of capsinoids, n ¼ 8) and controls (placebo, n ¼ 9). Diﬀerences (, y) where p < 0:05 or 0.1 were compared with the controls, while those (#, þ) where p < 0:05 or 0.1 were compared with the 0 week post-treatment values.

Table 3. Changes in Body Weight, BMI, SBP, DBP and Pulse Rate before and after Intake of Capsinoids

week 02 4 CSNs10 73:22 3:44 73:01 3:44 72:76 3:48 + Weight CSNs3 72:39 3:15 72:05 3:15 + 72:10 3:16 (kg) controls 73:44 3:19 73:35 Proofs3:10 73:42 3:08 CSNs10 26:18 0:76 26:10 0:76 26:01 0:78 + BMI CSNs3 26:02 0:64 25:90 0:65 + 25:92 0:65 (kg/m2) controls 26:10 0:87 26:07 0:81 26:09 0:81 CSNs10 110:67 2:58 113:07 3:65 113:33 3:58 SBP CSNs3 114:43 3:45 108:29 2:34 111:00 3:42 (mmHg) controls 112:40 2:32 114:27 2:60 114:00 2:17 CSNs10 73:73 2:49 73:07 2:72 74:80 2:55 DBP CSNs3 77:43 2:62 74:86 2:31 71:29 2:24 (mmHg) controls 77:20 2:63 76:27 2:31 74:27 1:55 CSNs10 66:53 2:40 69:07 1:69 71:47 2:13 + Pulse rate CSNs3 66:29 1:88 70:57 2:07 + 67:14 1:70 (bpm) controls 64:80 1:52 68:00 1:62 69:33 1:55

Each value is the mean SE. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure. CSNs10, n ¼ 15; CSNs3, n ¼ 14; controls, n ¼ 15. +: p < 0:1 vs. baseline by the Bonferroni test.

Discussion body temperature is inhibited by the TRPV1 antagonist, capsazepine.16) These results will indicate that capsi- The novel and much less pungent capsaicin ana- noids and capsaicin work via the TRPV1 receptor. logues, capsinoids, have an ester bond that replaces the TRPV1 is found on the surface of the oral cavity and amide bond linking the vanilloid group of capsaicin with digestive tract, so capsinoids may activate without being a fatty acid. The patch-clamp method has shown that absorbed into the peripheral circulation (unpublished ‘capsiate’ (one of the capsinoids) binds with the data). capsaicin receptor TRPV1.26) In addition, ‘capsiate’ In the present study, capsinoids were extracted from has been reported to increase the body temperature, ‘CH-19 sweet’ fruit and orally given at a dose of 3 or 15,16) enhance VO2 and decrease body fat. The increase in 10 mg/day for 4 consecutive weeks to investigate their Enhanced Energy Expenditure by Capsinoids in Humans 60341-7 Table 4. Average Daily Intake of Energy, Protein, Fat and Carbohydrate, Cholesterol, Fiber from Meals and Average Daily Number of Walking Steps before the Measurement Days

week 024 CSNs10 1863:5 76:1 1874:9 69:2 1872:1 54:5 Energy CSNs3 1877:6 52:4 1893:8 64:6 1867:1 49:4 (kcal/day) controls 1908:6 31:9 1874:5 64:8 1902:1 82:9 CSNs10 70:8 2:975:8 3:075:1 2:4 + Protein CSNs3 75:5 3:576:8 2:877:8 2:4 (g/day) controls 77:2 2:775:6 2:878:5 3:8 CSNs10 54:4 3:656:6 3:155:6 3:2 Fat CSNs3 59:9 3:754:2 3:257:1 2:8 (g/day) controls 61:4 1:757:4 2:861:9 3:2 CSNs10 261:6 10:4 253:7 9:1 256:6 7:8 Carbohydrate CSNs3 249:3 8:8 264:5 10:8 250:0 9:8 (g/day) controls 252:7 7:5 253:4 11:8 247:7 13:0 CSNs10 350:0 19:4 382:1 19:8 398:4 26:7 Cholesterol CSNs3 401:6 19:7 391:9 22:5 423:1 26:3 (mg/day) controls 412:5 27:3 443:7 34:0 430:2 33:6 CSNs10 11:8 0:611:4 0:611:5 0:6 Fiber CSNs3 13:0 1:113:5 1:213:1 1:1 (g/day) controls 13:4 0:712:3 0:712:6 0:9 AdvanceCSNs10 View11771:2 1322:2 11499:8 1294:5 11390:5 1044:2 Steps CSNs3 11019:9 1320:6 11149:6 1106:4 11436:5 1295:2 (steps/day) controls 12050:7 1367:9 11678:4 1452:8 11597:0 1209:9

Each value is the mean SE. CSNs10, n ¼ 15; CSNs3, n ¼ 14; controls, n ¼ 15.+:p < 0:1 vs. baseline by the Bonferroni test. effect on energy metabolism in humans. The results as UCP2 of white adipose tissue were demonstrated. disclosed a significant correlation between the increase Moreover, a single oral dose of ‘capsiate’ was shown to in fat oxidation and BMI. The increase in VO2 was elevate UCP1 mRNA of BAT and UCP3 mRNA of found to be significant, if a meta-analysis was made on skeletal muscle.17) Although BAT itself has been found 2 Proofs the subjects with BMI 25 kg/m . The increase in VO2 only in human neonates, UCP1 mRNA has been isolated could likely be attributable to the enhancement (either from human white adipose tissue that evidently con- significant or insignificant) of REE and fat oxidation, tained islets of brown adipocytes.27,28) As UCP2 ex- which were dose-dependently observable in this study. pression in fat tissue29) and UCP3 expression in skeletal In a previous animal study, a single oral adminis- muscles30) have been reported in humans, the UCP- tration of ‘capsiate’ increased VO2 following a 3-hr inducing activity of capsinoids may, at least in part, have period.15) A similar trend has been observed in previous contributed to enhancing the energy metabolism in this humans study using ‘CH-19 sweet’ fruits, where study. increased VO2 was maintained for at least 1 hr after Human BMR is generally measured immediately after oral administration.18) Contrary to the foregoing two waking up in the morning, with the subject resting in the studies, the respiratory gas was analyzed more than 24 hr supine position in a thermally comfortable environment after the last intake of capsinoids in the present study in without having eaten breakfast. The contents of the REE order to monitor the baseline elevation rather than the measured in the present study include not only basal acute response. This protocol was based on the previous metabolism, but unavoidable ‘metabolism due to other observation of mice treated with ‘capsiate’ for a 2-week factors.’ To standardize the ‘metabolism due to other period (10 mg/kg) displaying increased VO2 and fat factors’ among the subjects, they were asked to maintain oxidation for 24 hr even when measurements were their routine dietary habits, exercise, and dietary performed 24 hr after the final administration.17) The composition of meals. An increase in resting metabolism results of this study show that baseline elevation in VO2 observed in the present study could mainly have been seems less evident than acute one in human subjects. due to an increase in BMR. However, the increase in VO2 and tendency for Fat oxidation tended to be higher in CSNs3 and increasing REE and fat oxidation by the capsinoid CSNs10 than in the controls. Although there was no intake for 4 weeks strongly suggest that a long-term significant change in either the RQ, plasma TG or ketone treatment with capsinoids would enhance BMR. body levels, RQ in both CSNs3 and CSNs10 tended to In the animal experiment just referred to,17) increases be lower. Enhanced fat oxidation may contribute to in UCP1 protein and UCP1 mRNA of BAT as well increased energy expenditure. The increasing trend of 60341-8 N. INOUE et al. FFA level in CSNs10 is compatible with this notion, No significant changes in either diastolic and systolic because if fat oxidation in CSNs10 would have been blood pressure or pulse rate were apparent during the enhanced, lipolysis should have occurred beforehand test period. In addition, although significant changes which would induce an increase in the peripheral FFA were observed in certain hematological and biochemical level. Consistent with this notion, Masuda et al. have data, and blood-overall examination items, these reported previously that a 2-week continuous adminis- changes were within the normal range, and would not tration of ‘capsiate’ to mice enhanced fat oxidation in pose any significant clinical concern to a subject. Any the light phase (the resting period for mice) and induced adverse effects related to causal relationships of the 17) aVO2 increase. sample were not extracted in this study. A single oral administration of ‘capsiate’ to mice has In summary, a continuous 4-week intake of capsinoids been shown to increase the catecholamine, FFA and enhanced VO2 and tended to enhance REE and fat blood glucose levels concomitantly with a decrease in oxidation in subjects with obesity-categorized BMI TG level,15) and enhanced fat oxidation during running scores (25 kg/m2; according to the Japanese standard). exercise was augmented.31) These results suggest that an This implies that a capsinoid-induced change is an intake of capsinoids would influence fat metabolism important factor leading to possible weight reduction. such as lipolysis in fat tissues via the sympathetic Since the pungency of the doses used in this study was nervous system (SNS). Cumulative studies about the slight and the dose easy to be ingested, capsinoids are long-term effect of capsinoids on fat metabolism are thought to be potentially useful and applicable to many elucidating that enhanced metabolism may not be solely people. attributable to the elevation of UCP expression. 2 While BMI = 22 kg/m is designated as the lowest References criterion for the incidence of disease complications in Japan,Advance BMI 25 kg/m2 is categorized as obese. View BMI 1) Stunkard, A. J., Current views on obesity. Am. J. Med., has positive correlations with body fat, leptin level and 100, 230–236 (1996). PAI-1, and negative correlations with the adiponectin 2) Fricker, J., Rozen, R., Melchior, J. C., and Apfelbaum, and ghrelin levels.32–34) BMI is also correlated positively M., Energy-metabolism adaptation in obese adults on a with a decreasing rate of sleep energy expenditure.19) very-low-calorie diet. Am. J. Clin. 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